Cytochrome P-450-dependent metabolism in alveolar type II epithelial cells: modulation by platelet-activating factor.

Platelet-activating factor (PAF), an ether lipid mediator released from activated pulmonary phagocytes, was evaluated for its ability to affect cytochrome P-450-dependent activities in isolated rat alveolar type II cells. The data indicate that at non-toxic doses, PAF caused an increase in beta-naphthoflavone (BNF) inducible/alpha-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. At high concentrations of PAF, inhibition of both EtOPx'ase and metyrapone (MP) sensitive benzyloxyphenoxazone debenzylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAF analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF. PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in both intact cell and sonicated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type II cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.

Inhibition of stimulant-induced activation of phagocytic cells with tetrandrine.

Tetrandrine is an alkaloid obtained from the root of a medicinal herb which is employed in China as a treatment for silicosis. One proposed mechanism for the development of silica-induced fibrosis is lung damage resulting from particle-induced inflammation and secretion of reactive compounds from alveolar phagocytes. Therefore, the objective of the present study was to determine if tetrandrine exhibited the ability to inhibit respiratory burst activity of pulmonary phagocytes. The data indicate that although tetrandrine is not cytotoxic to phagocytic cells, it is a potent inhibitor in vitro of zymosan-stimulated oxygen consumption, superoxide anion release, and hydrogen peroxide secretion by alveolar macrophages. Tetrandrine is also effective in vivo in preventing activation of alveolar macrophages after inhalation or intratracheal instillation of silica. Tetrandrine also inhibits stimulant-induced chemiluminescence by polymorphonuclear leukocytes. Since tetrandrine does not alter stimulant-induced depolarization of phagocytic cells, its inhibitory action is not via interference with receptor-ligand binding but rather must occur elsewhere in the stimulus-secretion coupling scheme.

Acute silicosis responding to corticosteroid therapy.

The course of acute silicosis usually is relentlessly progressive. Death results from cor pulmonale and respiratory failure, with mycobacterial infection a frequent serious complication. Attempts to treat the illness generally have been unavailing. We report an unusual case of acute silicosis in which improvement in clinical status, chest x-ray film findings and pulmonary function occurred following therapy with corticosteroids. To our knowledge, this is the first such case reported in the medical literature.

Taurine uptake by isolated alveolar macrophages and type II cells.

Evidence suggests that taurine may protect cellular membranes against oxidants (Gordon et al., Am. J. Pathol. 125: 585-600, 1986). The present study was conducted to determine if alveolar macrophages and type II cells (which are relatively resistant to oxidant injury) possess a specialized transport system for the accumulation of taurine. The results indicate that both cell types contain more taurine than plasma or whole lung. Taurine influx exhibited both carrier-mediated and simple diffusion components. Carrier-mediated uptake displayed saturation kinetics (Km = 26.3 and 22.5 microM, while Vmax = 33.2 and 4.9 pmol.10(6) cells-1.min-1 for macrophages and type II cells, respectively). Taurine uptake was dependent on extracellular sodium and inhibited by metabolic inhibitors or ouabain. Total taurine uptake by type II cells was lower than that of alveolar macrophages. However, type II cells exhibited a higher intercellular concentration of taurine (14 vs. 4 mM) because of a higher ratio of carrier-mediated uptake to leakage than with alveolar macrophages. It is possible that this specialized transport system for taurine uptake may lend these cells resistant to oxidant injury.

Tripeptide analysis of collagen synthesized by silica-exposed fibroblast cultures.

The effect of silica on collagen biosynthesis by confluent monolayers of WI-38 fibroblast cultures was examined by a more comprehensive method of analysis. The presence of the particulates had no direct effect on protein (collagen) synthesis, proline incorporation or prolyl hydroxylase activity; the latter is determined by the degree of hydroxylation. Silica, however, was highly toxic to the cells.

Particle activity and in vivo pulmonary response to freshly milled and aged alpha-quartz.

This study examined the possibility of freshly fractured alpha-quartz being more toxic and inflammatory in vivo than aged quartz of the same composition and particle size. Fresh quartz was generated by a jet mill, and used immediately, while aged dust was stored for two months before use. Both the production of hydrogen peroxide and hydroxyl radicals and the analysis of surface radicals verified the enhanced surface activity of fresh quartz. Male Fischer 344 rats were exposed to fresh or aged alpha-quartz by inhalation (20 mg center dot m-3, 5 h per day, 5 d per week, for 2 weeks) and their pulmonary responses were determined 1--3 d postexposure. Exposure to aged quartz resulted in an increase in cytotoxic and inflammatory parameters. In comparison, the inhalation of freshly cleaved quartz resulted in dramatically greater increases in all of the pulmonary responses. This finding suggests that exposure to freshly machined quartz may result in a greater risk of pulmonary disease.

In vitro effects of straight-chain alkanes (n-hexane through n-dodecane) on rat liver and lung cytochrome P-450.

To evaluate the effect of straight-chain alkanes on normal detoxication reactions, we studied the in vitro effect of the homologous series n-hexane through n-dodecane on two cytochrome P-450 (EC 1.14.14.1) enzyme activities. Benzo[a]pyrene hydroxylase (BaPOHase) and 7-ethoxycoumarin deethylase activities were measured in liver and lung microsomes of control and beta-naphthoflavone-treated rats. In the presence of 2 mM n-hexane through n-dodecane, liver BaPOHase activity decreased from 67% of control with n-dodecane to 21% of control with octane. Lung benzo[a]pyrene hydroxylase was insensitive to all tested alkanes at 2 mM. In the presence of 2 mM alkanes, liver 7-ethoxycoumarin deethylase activity decreased from 73% of control with n-octane to 28% with n-octane. Lung 7-ethoxycoumarin deethylase was also sensitive to the alkane series. In the presence of 2 mM alkane the greatest effect was obtained with n-octane and represented a 56% loss in activity. Alkane concentration-dependence measurements showed 0.02-0.20 mM as the sensitive region of the curve for n-octane with maximal loss of activity achieved at 0.20 mM. Liver ethoxycoumarin deethylase activity from beta-naphthoflavone-treated rats was less sensitive towards the reactive alkane, n-octane, than the activity from control rats. Double-reciprocal-plot analysis revealed the maximal velocity (Vmax) was decreased in the presence of 0.2 mM n-octane. Hence this hydrocarbon did not exert its effect solely as an alternate substrate. The data show the n-alkanes, n-hexane through n-dodecane, interfered with a normal detoxication pathway in a manner that was chainlength-dependent, tissue-specific, and dependent on the preexposure history of the animal.

Platelet-activating factor-induced aggregation of rat alveolar macrophages.

Isolated rat alveolar macrophages aggregate in the presence of PAF in a dose- and cell-dependent manner. Saturation was achieved at 80 microM PAF. The response was increased linearly with cell number up to a concentration of 3 x 10(6) cells/ml, but decreased at higher cell concentrations. The stereoisomer, enantio-PAF, and the C2-acetyl hydrolyzed product, lyso-PAF, each caused aggregation of isolated rat alveolar macrophages in a manner similar to that for PAF. The PAF-induced aggregation of alveolar macrophages may be mediated through non-specific binding sites and may represent a toxic response to relatively high levels of PAF that released at localized sites.

In vitro effects of large and small glass fibers on rat alveolar macrophages.

The objective of this study was to explore the use of alveolar macrophage culture to evaluate the cytotoxicity of two glass fiber materials, a building insulation fiberglass (a relatively long and thick fiber) and a glass microfiber (a short and thin fiber). Alveolar macrophages were obtained from male Sprague-Dawley rats by bronchoalveolar lavage and were cultured with varying fiber concentrations for up to 3 d. Fiber toxicity was assessed by assaying cell viability, membrane integrity, and phagocyte function. The microfibers exhibited a concentration-dependent cytotoxicity shown by the loss of cell viability and function. The building insulation fiberglass had little effect on cell viability and did not change macrophage function in this assay system. The results of this study show that short and thin glass fibers are more toxic than long and thick fibers in vitro, supporting a role of fiber dimension in toxicity.

Lung cytochrome P450-dependent benzyloxyphenoxazone debenzylase and ethoxyphenoxazone deethylase activities in total microsomal and isolated alveolar type II cells: responses to changes in assay conditions with special reference to non-linear dependence at low enzyme concentration.

1. Responses of cytochrome P450-dependent ethoxyphenoxazone deethylase (EtOPx'ase) and benzyloxyphenoxazone debenzylase (BzOPx'ase) activities to changes in assay conditions were measured in total microsomal and isolated alveolar type II (tII) cells from rats pretreated with beta-naphthoflavone. 2. Whereas microsomal EtOPx'ase activity was unaffected by storage at -80 degrees C for up to 4 months, BzOPx'ase activity began to decline after only 1 month. 3. The microsomal and type II activities were unaffected by changes in pH (7.2-8.0) or salt content. 4. The type II activities increased after sonication 2.3-2.7-fold or in the presence of 10 microM dicumarol 1.7-1.9-fold. 5. Type II BzOPx'ase was sensitive to metyrapone (MP) whereas EtOPx'ase was sensitive to alpha-naphthoflavone (ANF). I50 values for the tII activities were calculated as: 0.63 microM--MP (BzOPx'ase), 80 microM--MP (EtOPx'ase), 0.024 microM--ANF (EtOPx'ase). At the highest concentration of ANF (10 microM), 50% inhibition tII BzOPx'ase was not observed. The results were similar to those obtained with the total lung microsomal fraction. 6. Microsomal and tII BzOPx'ase activities exhibited non-linear dependence at low enzyme concentration. Linearity was restored by 0.5 mM dimyristoylphosphatidylcholine.

Relative effects of asbestos and wollastonite on alveolar macrophages.

Rabbit alveolar macrophages were exposed in culture to chrysotile asbestos, wollastonite, or latex, and the effects on various biochemical and physiological parameters related to cellular viability and fibrogenicity were determined. Exposure of alveolar macrophages to asbestos, wollastonite, or latex for 3 d has no effect on oxygen consumption or cellular volume. However, treatment of alveolar macrophages with as little as 25 micrograms asbestos/ml for 1 d increases lysosomal enzyme release and decreases membrane integrity, i.e., decreases trypan blue exclusion and increases leakage of cytosolic enzymes. In contrast, exposure of alveolar macrophages to wollastonite or latex at 250 micrograms/ml does not induce lysosomal enzyme release or alter membrane integrity even after 3 d of exposure in culture. These data suggest that chrysotile asbestos damages rabbit alveolar macrophages, while wollastonite, a potential substitute for asbestos, is far less cytotoxic.

Taurine content of isolated rat alveolar type I cells.

1. Rat alveolar type I cells were isolated by enzymatic digestion and purified by centrifugal elutriation and specific surface adsorption. 2. The identity of the harvested cells was confirmed using electronic cell sizing and transmission electron microscopy. 3. Purified cell preparations contained 4.6 +/- 2.3 x 10(6) type I cells/rat lung with a purity of 79 +/- 3%. 4. Isolated type I cells exhibited the following characteristics: mean cell volume = 716 +/- 48 microns 3; diameter = 11.1 +/- 0.7 microns; and cell water content = 0.50 +/- 0.03 microliter/10(6) cells. 5. Taurine content of these alveolar type I cells was measured by HPLC. 6. The intracellular taurine concentration of type I cells was 0.14 +/- 0.07 mM, a value close to that of plasma (0.1 mM).

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity in rat alveolar type II cells: effect of pretreatment with beta-naphthoflavone.

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity was measured in alveolar type II cells from control and beta-naphthoflavone (ip) treated-rats. Type II cells were isolated from collagenase/elastase-digested lung tissue and purified by centrifugal elutriation. The specificity of the cytochrome P450-dependent activity towards four alkoxyphenoxazones (methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone) was measured under conditions that minimized interference by cytosolic conjugating- and NADPH-dependent quinone reductase activities. Ethoxyphenoxazone dealkylase activity was induced 17-fold following beta-naphthoflavone treatment and was further characterized by its kinetic parameters and sensitivities toward in vitro inhibitors (Km(app) = 0.20 microM, Vmax = 1.74 pmoles resorufin min-1 (10(6) cells)-1 10(6) cells; I50 (alpha-naphthoflavone) = 0.025 microM, and I50 (metyrapone) = 72 microM). beta-Naphthoflavone pretreatment of the rats did not result in statistically significant changes in methoxy-, pentoxy-, or benzyloxyphenoxazone dealkylase activity of alveolar type II cells, although, a trend towards decrease activity was observed for benzyloxyphenoxazone. beta-Naphthoflavone pretreatment had no effect on oxygen consumption or trypan blue exclusion in alveolar type II cells and macrophage ethoxyphenoxazone dealkylase and benzyloxphenoxazone dealkylase activities were not affected by the beta-naththoflavone pretreatment. The results show that exposure to beta-naphthoflavone resulted in an increase in type II cell cytochrome P450-dependent ethoxyphenoxazone dealkylase activity but not in other alveolar type II cell or macrophage alkoxyphenoxazone dealkylase activities or in parameters that monitor viability and cell wall integrity.